Octadecanoic acid, reaction products with diethylenetriamine, di-Me sulfate-quaternized

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

06 March 2018 - 13 April 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted 21st July 1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Octadecanoic acid, reaction products with diethylenetriamine, di-Me sulfate-quaternized

EC Number:

291-707-5

EC Name:

Octadecanoic acid, reaction products with diethylenetriamine, di-Me sulfate-quaternized

Cas Number:

90459-62-4

Molecular formula:

C24H55N3O6S

IUPAC Name:

bis(2-aminoethyl)amine octadecanoic acid dimethyl sulfate

Test material form:

solid

Remarks:

paste

Method

Target gene:

his

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

sodium phenobarbitone and β-naphthoflavone induced rat liver S9 fraction

Test concentrations with justification for top dose:

0.005, 0.016, 0.05, 0.16 and 0.5 mg/plate based on the results of solubility, precipitation and initial cytotoxicity test

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: distilled water

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

mitomycin C

other: 2-Aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DURATION
- Preincubation period: 30 min
- Exposure duration: 66 hours and 45 mins (plate incorporation), 65 hrs and 30 mins (preincubation assay)

NUMBER OF REPLICATIONS: 3

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 5

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: no precipitation

RANGE-FINDING/SCREENING STUDIES:
Based on the results of precipitation test, the concentrations of 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4 and 5 mg/plate were selected for the initial cytotoxicity test.
The test item resulted in cytotoxicity and it was observed by a thinning of the bacterial background lawn. The tester strain exposed with test item in the presence and absence of metabolic activation system resulted in cytotoxicity and those concentrations are graded as 0 (Absent lawn) for 0.8, 0.9, 1, 2, 3, 4 and 5 mg/plate, 1+ (extremely reduced lawn) for 0.7 mg/plate, 2+ (moderately reduced lawn) for 0.6 mg/plate and 3 + (Slightly reduced lawn) for 0.5 mg/plate when compared to vehicle control.
The test item was found to be cytotoxic up to the maximum concentration of 0.5 mg/plate tested when compared to the vehicle control.
On the basis of cytotoxicity results 0.5 mg/plate was considered as the highest test concentration for the main mutagenicity assay.

Any other information on results incl. tables

Solubility Test

The test item formed milky white suspension in distilled water at a concentration of 50 mg/mL.

 

Precipitation Test

The test item resulted in no precipitation at and up to 5 mg/plate.

 

Initial Cytotoxicity Test

Based on the results of precipitation test, the concentrations of 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4 and 5 mg/plate were selected for the initial cytotoxicity test.

The test item resulted in cytotoxicity and it was observed by a thinning of the bacterial background lawn. The tester strain exposed with test item in the presence and absence of metabolic activation system resulted in cytotoxicity and those concentrations are graded as 0 (Absent lawn) for 0.8, 0.9, 1, 2, 3, 4 and 5 mg/plate, 1+ (extremely reduced lawn) for 0.7 mg/plate, 2+ (moderately reduced lawn) for 0.6 mg/plate and 3 + (Slightly reduced lawn) for 0.5 mg/plate when compared to vehicle control.

The test item was found to be cytotoxic up to the maximum concentration of 0.5 mg/plate tested when compared to the vehicle control.

On the basis of cytotoxicity results 0.5 mg/plate was considered as the highest test concentration for the main mutagenicity assay.

 

TABLE 1.            SUMMARY OF INITIAL CYTOTOXICITY TEST-SALMONELLA TYPHIMURIUM TA100TESTER STRAIN

Test Item Concentration (mg/plate)	No. of Revertants/plate
	With S9						Without S9
	R1	R2	R3	Average	±SD	Bacterial Lawn Intensity	R1	R2	R3	Average	±SD	Bacterial Lawn Intensity
Vehicle Control	94	90	98	94	4.0	4+	99	95	92	95	3.5	4+
0.5	53	58	61	57	4.0	3+	50	55	52	52	2.5	3+
0.6	32	39	30	34	4.7	2+	33	36	29	33	3.5	2+
0.7	15	18	16	16	1.5	1+	17	19	15	17	2.0	1+
0.8	0	0	0	0	0.0	0	0	0	0	0	0.0	0
0.9	0	0	0	0	0.0	0	0	0	0	0	0.0	0
1	0	0	0	0	0.0	0	0	0	0	0	0.0	0
2	0	0	0	0	0.0	0	0	0	0	0	0.0	0
3	0	0	0	0	0.0	0	0	0	0	0	0.0	0
4	0	0	0	0	0.0	0	0	0	0	0	0.0	0
5	0	0	0	0	0.0	0	0	0	0	0	0.0	0

Values of Revertants are in Mean±SD

Lawn intensity:

4+= Thick lawn: Distinguished by a healthy (Normal) background lawn comparable to vehicle control plates.    

3+=Slightly thin lawn: Distinguished by a noticeable thinning (Slightly reduced) of the background lawn compared to vehicle control plates.

2+=Thin lawn: Distinguished by a marked thinning (Moderately reduced) of the background lawn compared to solvent/vehicle control plates.

1+=Very thin lawn: Distinguished by an extreme thinning(Extremely reduced)of the background lawn compared to solvent/vehicle control plates.

0=No lawn: Distinguished by a complete lack of any background lawn compared to solvent/vehicle control plates.

 

.    

 

TABLE 2.            SUMMARY OF COLONY COUNTS OF REVERTANTS OF TRIAL-I

          Plate Incorporation Method

                                                                                                                                         

Treatment	Test Concentration  (mg/plate)		No. of Revertants (Mean of 3 Plates)
			With S9						Without S9
			Salmonella typhimurium						Salmonella typhimurium
			TA 98	TA 100	TA 102	TA 1535	TA 1537		TA 98	TA 100	TA 102	TA 1535	TA 1537
Vehicle Control	100 µL of Distilled water	Mean	29.0	110.0	264.3	23.7	10.0		27.3	114.3	264.0	22.3	10.0
		±SD	3.6	5.6	12.9	2.5	3.6		3.2	22.0	7.9	3.1	2.0
	Lawn Intensity		4+	4+	4+	4+	4+		4+	4+	4+	4+	4+
Leomin KP	0.005	Mean	29.0	101.7	265.0	23.3	11.0		27.0	113.3	262.3	22.0	8.7
		±SD	4.0	4.7	11.4	2.5	1.0		3.0	21.1	5.5	2.0	3.8
	Fold Increase		1.0	0.9	1.0	1.0	1.1		1.0	1.0	1.0	1.0	0.9
	Lawn Intensity		4+	4+	4+	4+	4+		4+	4+	4+	4+	4+
	0.016	Mean	28.0	111.0	256.3	22.0	9.0		25.7	105.7	253.7	22.0	9.3
		±SD	2.0	10.6	8.1	3.0	2.6		3.5	8.1	6.4	3.6	0.6
	Fold Increase		1.0	1.0	1.0	0.9	0.9		0.9	0.9	1.0	1.0	0.9
	Lawn Intensity		4+	4+	4+	4+	4+		4+	4+	4+	4+	4+
	0.05	Mean	28.0	108.0	264.0	21.7	9.3		26.7	101.0	264.0	21.3	8.3
		±SD	4.0	6.0	11.5	3.2	2.1		2.5	10.6	5.0	2.5	3.2
	Fold Increase		1.0	1.0	1.0	0.9	0.9		1.0	0.9	1.0	1.0	0.8
	Lawn Intensity		4+	4+	4+	4+	4+		4+	4+	4+	4+	4+
	0.16	Mean	26.7	98.7	256.3	21.3	8.0		26.3	95.0	254.7	20.0	7.7
		±SD	4.9	8.0	11.2	2.5	1.0		3.2	7.2	4.7	3.0	1.5
	Fold Increase		0.9	0.9	1.0	0.9	0.8		1.0	0.8	1.0	0.9	0.8
	Lawn Intensity		4+	4+	4+	4+	4+		4+	4+	4+	4+	4+
	0.5	Mean	17.0	57.7	208.7	16.7	5.3		15.7	57.3	207.7	13.7	4.0
		±SD	2.6	4.0	7.5	0.6	3.2		1.5	5.0	6.7	1.5	1.0
	Fold Increase		0.6	0.5	0.8	0.7	0.5		0.6	0.5	0.8	0.6	0.4
	Lawn Intensity		3+	3+	3+	3+	3+		3+	3+	3+	3+	3+
Positive Control	100 µL of respective Positive Control	Mean	348.7	405.7	606.0	125.7	105.0		343.3	401.0	609.0	119.0	100.3
		±SD	15.7	15.3	7.0	8.5	4.6		18.4	10.5	8.2	14.9	4.2
	Fold Increase		12.0	3.7	2.3	5.3	10.5		12.6	3.5	2.3	5.3	10.0
	Lawn Intensity		4+	4+	4+	4+	4+		4+	4+	4+	4+	4+

Values of Revertants are in Mean±SD

Positive controls:

For with S9:

For Salmonella typhimurium TA98, TA100, TA102, TA1535 and TA1537 = 4 µg/plate of 2-Aminoanthracene

For without S9:

For TA98: 2 µg/plate of 2-Nitrofluorene

For TA100 and TA1535: 1 µg/plate of Sodium azide.

For TA102: 0.5 µg/plate of Mitomycin C

For TA1537: 50 µg/plate of 9-Aminoacridine

Applicant's summary and conclusion

Conclusions:

Based on the results of the study, it is concluded that the test item is non-mutagenic in the Bacterial Reverse Mutation Test up to the highest tested concentration of 0.5 mg/plate under the test conditions.

Executive summary:

In a reverse gene mutation assay in bacteria according to OECD Guideline 471, strains TA 1535, TA 1537, TA 98, TA 100 and TA 102 ofS. typhimurium were exposed toOctadecanoic acid, reaction products with diethylenetriamine, di-Me sulfate-quaternizedat concentrations of 0.005, 0.016, 0.05, 0.16 and 0.5 mg/plate based on the results of solubility, precipitation and initial cytotoxicity test in the presence and absence of mammalian metabolic activation (sodium phenobarbitone and β-naphthoflavone induced rat liver S9 fraction). Distilled water was used as vehicle. The test was performed as plate incorporation and pre-incubation assay.

Octadecanoic acid, reaction products with diethylenetriamine, di-Me sulfate-quaternized was tested uo to cytotoxic concentrations. The positive controls induced the appropriate responses.

There was noevidenceof induced mutant colonies over background.